Device for peeling cells

Abstract

To provide a device for peeling cells that solves the above problems by not only maintaining the position of a cell culture vessel but also making the position changeable.SOLUTION: Provided is a device for peeling cells that peels cells attached to the culture surface of a culture substrate by applying vibration to the culture surface, the device comprising: a vibration unit comprising a vibrator that generates the vibration, and a first holding unit that holds the culture substrate so that the horizontal position of the culture substrate relative to the vibrator can be changed.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a cell detachment device. [Background technology]

[0002] In the medical field, cells are sometimes cultured on the bottom of containers such as culture plates and dishes for treatment and research and development. However, adherent cells adhere to the bottom of the container. Therefore, it is necessary to detach and remove the adherent cells from the culture container.

[0003] Patent Document 1 discloses a cell separation device equipped with a detachment means for detaching a desired living cell from a living cell group from a wall surface of a container by irradiating the cell with ultrasonic waves.

[0004] Patent Document 2 discloses a vibration device that removes air bubbles generated during culture by vibrating a culture vessel. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP 2008-92857 A [Patent Document 2] JP 2015-43751 A Summary of the Invention [Problem to be solved by the invention]

[0006] The present inventors have found problems with the cell separation device and vibration device in the above-mentioned Patent Documents 1 and 2. That is, the cell separation device in Patent Document 1 does not have a configuration for fixing the container, and therefore, the position of the container may fluctuate due to vibrations generated by irradiation with ultrasonic waves, making it impossible to detach the cells.

[0007] In addition, the vibration device of Patent Document 2 discloses a configuration in which the culture vessel is held by applying a force in the vertical direction of the culture vessel. However, according to the study by the present inventors, it was found that the appropriate position for applying vibration changes in this configuration. That is, the peak position of the amplitude may change when vibration is continued to be applied, or the peak position of the amplitude may change when the vibration mode is changed. As a result, if the position of the culture vessel is held only at a specific position, it may be difficult to detach the cells, or the cells may be damaged, resulting in a decrease in the survival rate.

[0008] Therefore, an object of the present invention is to provide a cell detachment device that solves the above-mentioned problems by not only maintaining the position of a cell culture vessel but also making the position changeable. [Means for solving the problem]

[0009] The cell detachment device of the present invention is a cell detachment device that detaches cells attached to a culture surface of a culture substrate from the culture surface by applying vibration to the culture surface, and has a vibration unit including a vibrator that generates the vibration, and a first holding unit that holds the culture substrate so that the horizontal position of the culture substrate relative to the vibrator can be changed. Effect of the Invention

[0010] According to the cell detachment device of the present invention, the position of the culture vessel is not only maintained but also made changeable, so that the culture vessel is placed in an appropriate position and maintained in that position, and appropriate vibration is applied. As a result, it is difficult to detach the cells, and the survival rate of the cells is unlikely to decrease. [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 is a diagram showing a cell detachment device according to an embodiment of the present invention. [Diagram 2] FIG. 1 is a diagram showing a cell detachment device according to a first embodiment of the present invention. [Diagram 3] FIG. 13 is a diagram showing a cell detachment device according to a second embodiment of the present invention. [Figure 4] FIG. 13 is a diagram showing a cell detachment device according to a third embodiment of the present invention. [Diagram 5] FIG. 13 is a diagram showing a cell detachment device according to a fourth embodiment of the present invention. [Figure 6] FIG. 13 is a diagram showing a cell detachment device according to a fifth embodiment of the present invention. [Figure 7] 13A and 13B are diagrams showing examples of a weight in a cell detachment device according to a sixth embodiment of the present invention. [Figure 8] FIG. 13 is a diagram showing a cell detachment device according to a seventh embodiment of the present invention. [Figure 9] FIG. 13 is a diagram showing a cell detachment device according to an eighth embodiment of the present invention. [Figure 10] FIG. 13 is a diagram showing a cell detachment device according to a ninth embodiment of the present invention. [Figure 11] FIG. 13 is a diagram showing a cell detachment device according to a tenth embodiment of the present invention. [Figure 12] FIG. 23 is a diagram showing a cell detachment device according to an eleventh embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] A cell detachment device according to an embodiment of the present invention will be described with reference to Fig. 1. However, the present invention is not limited thereto. Fig. 1 is a schematic cross-sectional view of the cell detachment device according to this embodiment.

[0013] The cell detachment device 100 according to this embodiment detaches cells 3 attached to the culture surface of a culture substrate 8 from the culture surface by applying vibration to the culture surface. The cell detachment device 100 has a vibration unit 10 including an oscillator 16 that generates vibrations, and a first holder 1 that holds the culture substrate 8 so that the horizontal position of the culture substrate 8 relative to the oscillator 16 can be changed. In FIG. 1, the horizontal direction is the direction of a plane defined by the X-axis and the Y-axis, and the vertical direction is the Z-axis direction. The same applies to the cross-sectional views in FIG. 2 and subsequent figures.

[0014] Here, holding the horizontal position of the culture substrate 8 in a changeable manner means that the position of the culture substrate 8 can be changed without vibration being applied, and once the position change is complete, the position of the culture substrate 8 can be held (fixed) so that it does not fluctuate. By generating vibrations by the oscillator 16 while the culture substrate 8 is held, appropriate vibrations are applied to the culture substrate 8 and the cells 3 attached to its culture surface.

[0015] In the present embodiment, the first holding unit 1 is capable of changing the horizontal position of the culture substrate 8, so that the culture substrate 8 can be placed at any position on the vibration unit 10. For example, if the position at which the amplitude of the vibration generated by the vibrator 16 and transmitted to the culture substrate 8 is maximized changes, the first holding unit 1 can change the culture substrate 8 to an appropriate position at which the detachment rate and survival rate of the cells are increased.

[0016] The cell detachment device according to this embodiment may have a second holding unit 2 that holds the vertical position of the culture substrate. The second holding unit 2 may be a weight (loading means) or a pressure means that applies pressure to reduce the distance between the culture substrate and the vibrator. The weight may be configured to include a plurality of weights. The weight may be held by the first holding unit 1 as shown in FIG. 1, or may be suspended from the first holding unit 1. As shown in FIG. 1, the second holding unit may have a sieve portion 11 for engaging with the culture substrate 8. By having the sieve portion 11, it becomes easier to hold the position of the culture substrate 8. The first holding unit in this embodiment may have a rotation mechanism that can rotate around an axis perpendicular to the vibration surface of the vibrator, as described later. The rotation mechanism may have a vibration actuator that rotates a screw.

[0017] The cell detachment device according to this embodiment may have a position control unit 14 that controls the horizontal position of the culture substrate 8. The position control unit 14 is connected to a stage 41 that is movable in the XY directions to control the horizontal position of the culture substrate 8, and can control the movement. The position control unit 14 may be configured to control the rotation of the first holder 1 using the above-mentioned rotation mechanism.

[0018] The first holding part may be configured to be displaceable in the vertical direction. By being configured to be displaceable in the vertical direction, the first holding part can be retreated in the Z direction, or retreated in the Z direction and then retreated in the X and Y directions, which makes it easier to install and remove the culture substrate 8.

[0019] The culture substrate 8 in this embodiment may include a container 7 having a cell culture surface and an opening, and a lid 6 that covers the opening. The first holder 1 may be configured to hold the lid 6 as shown in FIG. 1, or may be configured to hold the container 7.

[0020] The cell detachment device according to this embodiment may have an acoustic transmission medium 9 between the vibration unit 10 and the culture substrate 8. The acoustic transmission medium is provided in a path for transmitting vibrations, particularly ultrasonic band vibrations, from the vibration unit 10 to the culture substrate 8 so as not to interpose an air layer that would cause vibration attenuation. The acoustic transmission medium may be a solid such as silicone rubber or a liquid such as water. The acoustic transmission medium may be a liquid, and the first holder may be configured so that the culture substrate is in contact with the acoustic transmission medium and the culture substrate is not in contact with the vibration unit.

[0021] In addition to the transducer 16, the vibration section 10 in this embodiment may include a vibration plate 15 for transmitting the vibration generated by the transducer 16. The vibration generated by the transducer 16 may be in the ultrasonic band.

[0022] An example of a cell detachment device according to an embodiment of the present invention will be described below.

[0023] First Embodiment An example of a cell detachment device according to a first embodiment of the present invention is shown in FIG.

[0024] The cell detachment device according to this embodiment is provided on a culture substrate 8, and irradiates the cultured cells 3 with ultrasonic vibrations (hereinafter sometimes abbreviated as ultrasonic vibrations), thereby detaching the cells 3 from the culture substrate 8. The cell detachment device 100 has a disk-shaped vibration section 10 including a piezoelectric element (vibrator) 16 fixed to a vibration plate 15 (not shown). The piezoelectric element 16 is provided with electrodes (not shown) for applying an alternating signal, and the vibration section 10 is supported by a bottom plate 12. The piezoelectric element 16 is provided with electrodes (not shown) on the front and back sides with opposite polarities to cause stretching vibration, and a folded electrode is also provided on the front side to enable electrical conduction between the back electrode and the front side. This configuration allows ultrasonic vibrations to be excited and irradiated by applying an alternating signal to the piezoelectric material.

[0025] In the cell detachment device according to the present embodiment, ultrasonic vibration is transmitted and irradiated from the vibration plate 15 of the disk-shaped vibration unit 10 to the culture substrate 8. However, as shown in FIG. 2(b), the surfaces of the culture substrate 8 and the vibration plate 15 are not necessarily flat and may be slightly deformed. In such a case, the vibration may be difficult to transmit. Therefore, in order to bring the bottom surface of the container 7 into even contact with the vibration plate 15, the center of gravity of the weight (second holding unit) 2 and the center of gravity of the culture substrate 8 are aligned as shown in FIG. 2(c), so that the weight 2 is weighted in a balanced manner against the culture substrate 8. If there is a gap between the vibration unit 10 and the culture substrate 8, the ultrasonic vibration may be attenuated in the gap and the necessary vibration may not be obtained, so appropriate weighting by the weight 2 is necessary. Conversely, if the weight 2 is too heavy, it may hinder the vibration of the vibration unit 10, so it is preferable to select a weight suitable for the container 7.

[0026] Here, when ultrasonic vibrations are applied to the culture substrate 8, the culture substrate 8 floats up together with the weight 2 by a small amount of 10 μm or less due to the irradiation pressure of the ultrasonic vibrations, and the position becomes unstable and may shift from the initial position. As a means for determining the position of the culture substrate 8, a vibration unit 10, a weight 2 that applies weight vertically to the culture substrate 8 provided on the vibration unit 10, and a first holding unit 1 that positions the culture substrate 8 and the weight 2 to hold the positions relative to the transducer 16 are provided. The positioning tool 1 in FIG. 2 is an example of the first holding unit 1. With this configuration, it is possible to reduce deviation of the positional relationship between the culture substrate 8 on which the cells are placed and the transducer 16 from the initial setting.

[0027] The configuration of the cell detachment device according to the embodiment will be specifically described. First, the configuration of the substrate 8 including the culture vessel 7. The cells 3 are cultured in the culture substrate 8 including the vessel 7 and the lid 6 covering the opening of the vessel 7, and are in a state of being adhered to the bottom of the vessel 7. The lid 6 prevents contaminants from coming into contact with the cells 3, that is, to reduce contamination. The culture substrate 8 is preferably configured so that the lid 6 fits into the vessel 7. In addition, by providing the lid 6, contamination caused by friction debris and the like caused by contact with the weight 2 integrated with the positioning tool 1 when automatically opening and placing the weight 2, as described later, can be reduced.

[0028] The vibration unit 10 in the cell detachment device 100 according to this embodiment has a shape that allows the culture substrate 8 to be placed on the upper surface of the vibration unit 10. For example, if the bottom of the container is flat, the vibration surface of the vibration unit 10 is similarly flat. A vibration transmission medium for transmitting vibrations more efficiently may be provided between the vibration surface and the culture substrate 8. The vibration transmission medium may be a liquid or gel such as water or glycol, or a solid such as silicone rubber. Rubber is an elastic material that easily deforms and adheres to the surface, and can be used as the acoustic transmission member.

[0029] As mentioned above, the vibration surface of the vibration unit 10 and the bottom surface of the container 7 are not necessarily flat and may be slightly deformed, so that any gaps between them make it difficult for vibration to be transmitted. Therefore, in order to bring the bottom surface of the culture substrate 8 into contact with the vibration surface evenly, the weight 2 is applied with a force appropriate for the deformation of each substrate. It is desirable that the surface where the weight 2 comes into contact with the substrate 8 is also flat so that the weight 2 acts on the culture substrate 8 with an even pressure.

[0030] In this embodiment, there is provided a weight 2 as a second holder, and a positioning tool 1 (first holder) that indicates a changeable position of the culture vessel while holding the weight 2. The positioning tool 1 can be configured not to constrain the axis in the gravity direction.

[0031] Examples of the positioning tool 1 are shown in Figs. 1 and 2. In Fig. 1, the positioning tool 1 is fixed to a stage 4 that can move in the horizontal direction. The stage 4 allows the culture substrate 8 on which the cells 3 have been cultured to be placed on the vibration unit 10 so that the position is small when vibration is applied. The appropriate position is a position where a force acts on the adhesion interface of the cells 3, and may change over time depending on the progress of detachment, or may change due to the vibration mode generated by the vibrator 16 or the aging of the vibrator 16 itself. In addition, since the strength of irradiation appears depending on the shape of the vibration transmission medium 9 and the shape of the vibration plate 15, it is extremely important that the culture substrate 8 can be moved to an appropriate position when vibration is applied. For this reason, it is preferable to have a position control unit 14 that has a stage control circuit for controlling the stage 4 and electrically controls the position of the stage to change the position of the culture substrate 8. It is also possible to use a switching unit 13 to switch between the operation of applying the vibration unit 10 and the operation of changing the position of the culture substrate 8 using the first holding unit 1.

[0032] Second Embodiment An example of a cell detachment device according to a second embodiment of the present invention is shown in FIG.

[0033] In a configuration in which the weight 2 (second holding part) is supported by being fitted with the positioning tool 1 (first holding part), a retraction process of the positioning tool 1 and a process of attaching and detaching the weight 2 are required. In the configuration according to this embodiment, even in the case of the weight 2 that does not restrain the axis in the direction of gravity, the positioning tool 1 and the weight 2 are configured to be floating in the air and integrated. FIG. 6(a) shows a state in which the weight 2 is suspended from the positioning tool 1 via a hanging member 21 such as a wire (reinforced fiber, metal wire, piano wire, etc.). In FIG. 6(b), the slide shaft 22 is fixed to the weight 2 and fitted with the positioning tool 1, and the weight 2 is suspended as a structure that slides with a ball bearing, a metal bearing, etc. The weight 2 is movable up and down, and can be prevented from falling by a stop ring 23. The slide shaft 22 may be fixed to either the positioning tool 1 or the weight 2. The hanging member 21 may be a flexible sheet-like material such as rubber (FIG. 6(c)).

[0034] In addition, as shown in Fig. 15, the positioning tool 1 and the weight 2 can be retracted with a single operation, making it easy to attach and detach the culture substrate 8. The front and back states of the positioning tool 1 rotated around the rotating support 18 are shown in Fig. 6(d) and (e).

[0035] The weight 2 as the second holding part is hung by a string or the like from the positioning tool 1 as the holding part, thereby obtaining the effect of equalizing the load. Since the load is applied by gravity, it is stable, and this solves the problem that it is difficult to create a stable equalized state of pressure due to the shape of the elastic body such as the clamping spring and the settling of the elastic body as disclosed in Patent Document 2.

[0036] Third embodiment An example of a cell detachment device according to a third embodiment of the present invention is shown in FIG.

[0037] FIG. 4 is a schematic cross-sectional view of the cell peeling device according to the present embodiment.

[0038] A support part 5 and a stage 4 are installed on the bottom plate 12. A vibration part 10 including a vibration plate 15 and a piezoelectric element 16 (vibrator) is attached to the support part 5. The culture substrate 8 is placed on the upper surface of the vibration plate 15, and the weight 2 (second holding part) is stacked in a balanced manner on the upper surface of the substrate shown in the figure so that the culture substrate 8 is unlikely to tilt. As shown in FIG. 4(b), the relationship between the weight 2 and the culture substrate 8 is preferably such that the center of gravity of the culture substrate 8 and the center of gravity of the weight 2 are approximately aligned with the direction of gravity (the normal direction of the vibration plate 15). FIG. 4(c)(c) shows an example of a poor balance, where the pressure is not uniform and the weights are not parallel. The weight 2 is not limited to a circular shape and may have other shapes. For example, FIG. 4(e) shows an example of a rectangular weight 2.

[0039] In this embodiment, as shown in FIG. 4, a fitting portion 11 is provided to reduce misalignment and maintain balance, so that a plurality of weights can be selectively stacked.

[0040] (Fourth embodiment) An example of a cell detachment device according to a fourth embodiment of the present invention is shown in FIG.

[0041] 5 is a top view of the cell detachment device according to this embodiment.

[0042] A culture substrate 8 is provided in the center of the support part 5, and a weight 2 is placed on the culture substrate 8 and engaged with the positioning tool 1. A stage 4 is provided on a bottom plate 12, and the positioning tool 1 is fixed thereto. By moving the stage 4, the positioning tool 1 becomes freely movable in a direction parallel to the vibration plane of the vibration part 10 as shown in the figure, and the culture substrate 8 is also freely movable via the weight 2.

[0043] Fifth embodiment An example of a cell detachment device according to a fifth embodiment of the present invention is shown in FIG.

[0044] In this embodiment, the positioning tool 1 is incorporated into the weight 2 having the engagement portion 11 with the culture substrate 8.

[0045] As shown in Fig. 6(a), the positioning tool 1 is a cylindrical pin-shaped tool that is evenly arranged in three positions. The outer diameter of the cylinder is connected to a circle so that the diameter fits the inner diameter of the vibration tank 26 of the cell detachment device shown in Fig. 6(c). This provides the same effect as the second embodiment. As shown in Fig. 6(b), the positioning tool 1 and weight 2 can be retracted in the normal direction (in the figure) with a single operation, making it easy to attach and detach the culture substrate 8.

[0046] (Sixth embodiment) An example of a cell detachment device according to a sixth embodiment of the present invention is shown in FIG.

[0047] Figure 7(a) shows the state when weights 2 are stacked in two layers, and Figure 7(b) shows a configuration with more than two layers of weights. As shown in Figure 7(c), weights 2 and 2d may have different shapes. When stacking multiple weights 2, the lower height may be more stable, in which case Figure 7(c) should be used.

[0048] 7(d) shows a form adapted to the second embodiment. In this configuration, weight 2a is suspended from positioning tool 1 by slide shaft 22, and a retaining ring 23 is provided at the upper end of the slide shaft to prevent it from falling off. At this time, weights 2b and 2c can be stacked on weight 2a.

[0049] Seventh embodiment An example of a cell detachment device according to a seventh embodiment of the present invention is shown in FIG.

[0050] Here, only the points that differ from the first embodiment of the present invention will be described.

[0051] The cell detachment device according to this embodiment is equipped with a screw 24 and a friction member 25 on the support part 5, and is provided with a rotation stopper 17. The mounting tool 1 is incorporated into the screw and rotates together with the rotation of the screw. When the screw rotates up to the rotation stopper 17, the rotation stops, and the mounting tool 1 rises or falls in the same way as a normal screw.

[0052] 8(a)(b) are a side cross-sectional view and a top view of the positioning tool 1 in a retracted state. When the screw rod is rotated counterclockwise during retraction, it rises to the position where the lid 6 of the culture substrate 8 is hung. After that, it leaves the culture substrate 8 and moves to the retracted position, allowing it to be raised to any position. Also, FIG. 8(c)(d) shows a state in which the weight 2 and the culture substrate 8 are installed so that they can be engaged. When the screw rod is rotated in the reverse direction, it reaches the set position, hits the rotation stopper, and drops. The friction member 25 is a member that applies a load that adjusts the rotation resistance, and it is desirable to provide a certain degree of resistance to the rotating side so that the positioning tool 1 can rotate stably during rotation. For example, if the load is light, the positioning tool will be pushed back to the other side due to the impact of hitting the rotation stopper, so it is preferable to provide such a friction member.

[0053] Eighth embodiment An example of a cell detachment device according to an eighth embodiment of the present invention is shown in FIG.

[0054] Regarding the cell detachment device according to this embodiment, only the points that differ from the seventh embodiment of the present invention will be described.

[0055] In this embodiment, a rotation actuator 28, which is a part of the rotation mechanism (screw 24, friction member 25, rotation actuator 28), is provided to automate the movement of the culture substrate by the first holder. As shown in the figure, the rotation actuator can rotate forward and backward, and can be controlled by a position control unit 14 shown in the figure.

[0056] Ninth embodiment An example of a cell detachment device according to a ninth embodiment of the present invention is shown in FIG.

[0057] In this embodiment, the vibration unit 10 is configured to generate ultrasonic vibrations as shown in the figure. The device is equipped with a disk-shaped vibration section 10 having a piezoelectric element 16 fixed to a vibration plate 15. The piezoelectric element 16 has an electrode (not shown) for applying an alternating signal, and the vibration section 10 is supported by a support section 5. The piezoelectric element 16 has electrodes (not shown) on the front and back sides with opposite polarity to cause stretching vibration. A folded electrode is also provided on the front side to allow electrical conduction between the back electrode and the front side. By applying an alternating signal to the piezoelectric material, ultrasonic vibration can be excited and emitted. The piezoelectric element does not have to be a ring resonator as shown in Figure 10, and various shapes can be made using various transducers such as Langevin type, surface acoustic wave type, and laminate type. When ultrasonic vibration is used, the container becomes easier to move as shown in the figure.

[0058] (Tenth embodiment) An example of a cell detachment device according to a tenth embodiment of the present invention is shown in FIG.

[0059] In the cell detachment device according to this embodiment, the contact portion of the weight 2 is a uniform flat surface as shown in the figure, and as shown in Fig. 11(b), the weight is shaped so that the center of gravity of the weight 2 substantially coincides with the center of the projected upper surface of the culture substrate 8. To achieve more uniform contact without gaps, it is also effective to interpose a soft elastic material such as rubber on the flat surface.

[0060] (Eleventh embodiment) An example of a cell detachment device according to an eleventh embodiment of the present invention is shown in FIG.

[0061] The following describes the differences from the configuration of the cell detachment device according to the embodiment of the present invention described with reference to FIG. 1. In this embodiment, the acoustic transmission medium 9, which is a liquid, is held by the holding unit 30 of the acoustic transmission medium 9, the culture substrate 8 is in contact with the acoustic transmission medium 9, and the culture substrate 8 is not in contact with the vibration unit 10. The first holding unit 1 holds the culture substrate 8, and the position at which the culture substrate 8 is held can be changed by using, for example, a pin with a variable length as the first holding unit 1. As shown in FIG. 12, the third holding unit 29 may be configured to more easily hold the position of the culture substrate 8. The third holding unit 29 may be made of, for example, a mesh-like material, and the culture substrate is placed on the mesh-like material and placed in the acoustic transmission member 9, which is a liquid, to easily hold the position of the culture substrate 8 while bringing the acoustic transmission medium 9 into contact with the culture substrate 8. [Explanation of symbols]

[0062] 1 First holding part 2 Second holding part 3 cells 4 Stage 5 Support part 6 Lid 7 containers 8 Culture substrate 9. Acoustic Transmission Media 10 Vibration section 11 Engagement part 12 Bottom plate 13 Switching section (switch box) 14 Position control section 15 Vibration plate 16 Transducers 17 Anti-rotation 18 Rotating Support 19 Support member 20 Fixtures 21 Suspension members 22 Slide shaft 23 Retaining ring 24 Screw 25 Friction materials 26 Vibration tank 27 Rotating mechanism 28 Rotary Actuator 29 Third Retaining Part 30 Acoustic transmission medium holder 100 Cell detachment device

Claims

1. A cell detachment device that detaches cells attached to a culture surface of a culture substrate from the culture surface by applying vibration to the culture surface, a vibration unit including a vibrator that generates the vibration; a first holding section that holds the culture substrate so that a horizontal position of the culture substrate relative to the transducer can be changed;

2. The cell detachment device according to claim 1 , further comprising a second holding portion for holding the vertical position of the culture substrate.

3. The cell detachment device according to claim 2 , wherein the second holding portion is a weight.

4. The cell detachment device according to claim 3 , wherein the weight comprises a plurality of weights.

5. The cell detachment device according to claim 3 , wherein the weight is suspended from the first holding portion.

6. The cell detachment device according to claim 2 , wherein the second holding unit has a pressure applying means for applying pressure so as to reduce the distance between the culture substrate and the transducer.

7. The cell detachment device according to claim 1 , wherein the first holding portion has an engagement portion for engaging with the culture substrate.

8. The cell detachment device according to claim 1 , further comprising a rotation mechanism capable of rotating the first holding part about an axis perpendicular to a vibration plane of the transducer.

9. The cell detachment device according to claim 8 , wherein the rotation mechanism includes a vibration actuator that rotates a screw.

10. The cell detachment device according to claim 1 , further comprising a position control unit for controlling a horizontal position of the culture substrate relative to the transducer.

11. The cell detachment device of claim 10, further comprising a rotation mechanism that can rotate the first holding unit around an axis perpendicular to the vibration plane of the transducer, and the position control unit uses the rotation mechanism to control the rotation of the first holding unit.

12. The cell detachment device according to claim 1 , wherein the first holding portion is configured to be displaceable in a vertical direction.

13. The cell detachment device according to claim 1 , wherein the culture substrate includes a container having a culture surface for the cells and an opening, and a lid for covering the opening.

14. The cell detachment device according to claim 13 , wherein the first holding portion holds the lid.

15. The cell detachment apparatus according to claim 13 , wherein the first holding portion holds the container.

16. The cell detachment device according to claim 1 , further comprising an acoustic transmission medium between the vibration unit and the culture substrate.

17. the acoustic transmission medium is a liquid; The cell detachment device according to claim 16 , wherein the first holding unit is configured so that the culture substrate is in contact with the acoustic transmission medium and the culture substrate is not in contact with the vibration unit.

18. The cell detachment device according to claim 1 , wherein the transducer generates vibrations in the ultrasonic band.

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